SOIL PROTOZOA 323 



travel faster than bacteria is utilized in the making of transfers. Bad. 

 fluorescens was used to repress the accompanying bacteria. 



Staining of protozoal Whenever possible, protozoa should be ex- 

 amined in a living condition, by suspending a drop of the medium or a 

 suspension in water in a hanging drop. The motility of protozoa can 

 be reduced as pointed out elsewhere (p. 45); various colloids (e.g. 

 5 per cent gelatin solution), narcotics (e. g. cocaine) or other poisons in 

 subletal concentrations (e.g. tannic acid) can be used, varying with the 

 species. Among the vital stains, 1 to 800-10,000 neutral red, 1 to 10,000 

 -100,000 Bismark brown, nigrosin, methylene blue, malachite green or 

 1 to 500,000 cyanin may be used. 



For the examination of finer structure, the protozoa are first fixed, while 

 moist, then stained and finally dehydrated without allowing the preparation to 

 dry. Mayer's albumin (50 cc. filtered egg-white + 50 cc. glycerol, + 1 gram 

 sodium salicilate) may be first spread over the slide to prevent the washing away 

 of the protozoa. A number of fixative agents have been suggested; of these a 

 few may be mentioned: 



a. Sublimate fixative (Schaudinn's): 2 parts of a concentrated aqueous solu- 

 tion of sublimate + 1 part of 96 per cent alcohol + 5 per cent acetic acid. Fix 

 10 to 30 minutes, wash in 60 per cent alcohol containing some iodine and potassium 

 iodide, then in 70 per cent solution. 



b. Picric acetic acid: 95 cc. of concentrated aqueous solution of picric acid 

 + 5 cc. of acetic acid. Fix for 10 minutes, then wash with 50 per cent alcohol, 

 then in 70 per cent alcohol. 



c. Chromo-aceto-osmic acid (Flemming's): 15 cc. of 1 per cent chromic acid, 

 4 cc. of 2 per cent osmic acid, and 1 cc. of acetic acid. Fix for 30 minutes and 

 wash with water. 



d. Picro-formol: 75 parts of an aqueous saturated picric acid solution, 25 parts 

 of formaldehyde, 5 parts of acetic acid. 



For staining purposes a number of dyes have been recommended, a few of 

 which are given here: 



a. Heidenhain's iron haemotoxylin. The preparation is first treated with 3 

 per cent solution of ferric alum (NH4) 2 Fe 2 (S0 4 )4, for 30 minutes, then washed 

 with water and placed in a 0.5 per cent aqueous solution of haemotoxylin for 1 to 

 14 hours, then washed with water and treated with 1.5 per cent solution of ferric 

 alum. As soon as a satisfactory differentiation has been obtained, the prepara- 

 tion is washed for 30 minutes in flowing water. 



44 A detailed review of the fixing and staining or protozoa is given by Hargitt, 

 Doflein and von Prowazek, while the staining of soil protozoa is given by Goodey. 

 Hargitt, G. W. Methods of studying and mounting protozoa. Jour. Appl. 

 Micros., 385-388. 1899; Goodey, T. A contribution to our knowledge of the 

 protozoa of the soil. Proc. Roy. Soc. (London) B, 84: 165-180. 1911; 88: 437- 

 456. 1915; 89: 297-314. 1916. See also Bolles Lee. The microtomist's Vade 

 mecum, and N. Hartmann. Praktikum der Protozoologie. Fischer. Jena. 



